pedoman assay kit no ka1342

8/20/2019 pedoman assay kit NO KA1342

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www.abnova.com

Nitrate/Nitrite Colorimetric

Assay Kit

Catalog Number KA1342

96 assays

Version: 06

Intended for research use only


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Table of Contents

Introduction ...................................................................................................... 3

Background ................................................................................................................... 3

Principle of the Assay .................................................................................................... 4

General Information ......................................................................................... 6

Materials Supplied ......................................................................................................... 6

Storage Instruction ........................................................................................................ 6

Materials Required but Not Supplied ............................................................................. 6

Precautions for Use ....................................................................................................... 7

Assay Protocol ................................................................................................. 8

Reagent Preparation ..................................................................................................... 8

Sample Preparation ....................................................................................................... 9

Assay Procedure ........................................................................................................... 9

Data Analysis.................................................................................................. 13

Calculation of Results .................................................................................................. 13

Performance Characteristics ....................................................................................... 14

Resources ....................................................................................................... 15

Troubleshooting ........................................................................................................... 15

References .................................................................................................................. 15

Plate Layout ................................................................................................................ 16


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Introduction

Background

Nitric Oxide (NO) is synthesized in biological systems by the enzyme Nitric Oxide Synthase (NOS). NOS is a

remarkably complex enzyme which acts on molecular oxygen, arginine, and NADPH to produce NO, citrulline,

and NADP+. This process requires five additional cofactors (FMN, FAD, Heme, calmodulin, and

tetrahydrobiopterin) and two divalent cations (calcium and heme iron; see Figure 1). Three distinct isoforms of

NOS have been identified, as detailed in Figure 2.

Figure 1. Nitric oxide synthesis

Figure 2. Nitric oxide synthase isoforms


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NO is produced in trace quantities by neurons, endothelial cells, platelets, and neutrophils in response to

homeostatic stimuli.1,2

This NO is scavenged rapidly (t1/2 = 4 seconds) and acts in a paracrine fashion to

transduce cellular signals. NO interacts with the heme prosthetic group of guanylate cyclase, activating the

enzyme and leading to increased cGMP levels. NO is also produced by other cells (macrophages, fibroblasts,

hepatocytes) in micromolar concentrations in response to inflammatory or mitogenic stimuli. In this case, the

biological role is defense against non-self pathogens through oxidative toxicity. These very high NO levels lead

to the formation of peroxynitrite, destruction of iron-sulfur clusters, thiol nitrosation, and nitration of protein

tyrosine residues. Thus, the amount of NO produced in different biological systems can vary over several

orders of magnitude and its subsequent chemical reactivity is diverse.

NO undergoes a series of reactions with several molecules present in biological fluids. These include:

The final products of NO in vivo are nitrite (NO2-) and nitrate (NO3

-). The relative proportion of NO2

- and NO3

- is

variable and cannot be predicted with certainty. Thus, the best index of total NO production is the sum of both

NO2- and NO3

-.

Principle of the Assay

Nitrate/Nitrite Colorimetric Assay Kit provides an accurate and convenient method for measurement of total

nitrate/nitrite concentration in a simple two-step process. The first step is the conversion of nitrate to nitrite

utilizing nitrate reductase. The second step is the addition of the Griess Reagents which convert nitrite into a

deep purple azo compound (See Figure 3).3 Photometric measurement of the absorbance due to this azo

chromophore accurately determines NO2- concentration.

Figure 3. Chemistry of the Griess Reagents


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NADPH is an essential cofactor for the function of the NOS enzyme. Unfortunately, NADPH interferes with the

chemistry of the Griess reagents, which are the most commonly used reagents for nitrite detection. One way to

prevent this interference is to use small amounts of NADPH in conjunction with a catalytic system for recycling

spent NADP+ back to NADPH. This is the system used in this Nitrate/Nitrite Colorimetric Assay Kit. It works

well for the analysis of nitrate and nitrite in fluids such as plasma and urine, and is also available in a highly

sensitive fluorometric version for the detection of low levels of nitrite. However, it cannot be used to analyze

nitrate and nitrite from an in vitro assay of NOS in which excess NADPH has been added. For these assays a

second method (LDH method) is utilized in a 96-well plate format.


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General Information

Materials Supplied

List of component

Item Quantity/Sizes

Nitrate/Nitrite Assay Buffer 1 vial

Nitrate Reductase Enzyme Preparation 2 vials

Nitrate Reductase Cofactor Preparation 2 vials

Nitrate Standard 1 vial

Nitrite Standard 1 vial

Griess Reagent R1 2 vials

Griess Reagent R2 2 vials

96-Well Solid Plate (Colorimetric Assay) 3 plates

96-Well Cover Sheet 3 covers

Storage Instruction

This kit will perform as specified if stored as directed at -20°C and used before the expiration date indicated on

the outside of the box.

Item Storage

Nitrate/Nitrite Assay Buffer 4°C

Nitrate Reductase Enzyme Preparation -20°C

Nitrate Reductase Cofactor Preparation -20°C

Nitrate Standard 4°C

Nitrite Standard 4°C

Griess Reagent R1 4°C

Griess Reagent R2 4°C

96-Well Plate (Colorimetric Assay) Room Temperature

96-Well Cover Sheet Room Temperature

Materials Required but Not Supplied

A plate reader capable of measuring absorbance at 540-550 nm

Adjustable pipettes and a repeat pipettor

A source of pure water (preferably Milli-Q or equivalent)


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Precautions for Use

Warning: This product is for laboratory research use only: not for administration to humans. Not for human or

veterinary diagnostic or therapeutic use.

Pipetting Hints

It is recommended that an adjustable pipette be used to deliver reagents to the wells.

Before pipetting each reagent, equilibrate the pipette tip in that reagent (i.e., slowly fill the tip and gently

expel the contents, repeat several times).

Do not expose the pipette tip to the reagent(s) already in the well.


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Assay Protocol

Reagent Preparation

Nitrate/Nitrite Assay Buffer - Dilute the contents of the Assay Buffer vial to 100 mL with UltraPure water

(Milli-Q or equivalent). This Assay Buffer should be used for dilution of samples as needed prior to assay.

The buffer will be stable for approximately two months at 4°C.

Nitrate Reductase Enzyme Preparation - Reconstitute the contents of the vial with 1.2 mL of Assay Buffer.

Keep on ice during use. Store at -20°C when not in use. Freezing and thawing of this solution should be

limited to one time.

Nitrate Reductase Cofactors Preparation - Reconstitute the contents of the vial with 1.2 mL of Assay

Buffer. Keep on ice during use. Store at -20°C when not in use. Freezing and thawing of this solution

should be limited to one time. Nitrate Standard - Remove the vial stopper slowly to minimize disturbance of the lyophilized powder.

Reconstitute the contents of the vial with 1.0 mL of Assay Buffer. Vortex and mix sufficiently to ensure all

powder in the vial, including any on the stopper, is in solution. Store at 4°C when not in use (do not

freeze!). The reconstituted standard will be stable for about four months when stored at 4°C.*

Nitrite Standard - Remove the vial stopper slowly to minimize disturbance of the lyophilized powder.

Reconstitute the contents of the vial with 1.0 mL of Assay Buffer. Vortex and mix sufficiently to ensure all

powder in the vial, including any on the stopper, is in solution. Store at 4°C when not in use (do not

freeze!). The reconstituted standard will be stable for about four months when stored at 4°C.*

Griess Reagents R1 and R2 - Do not add any water or Assay Buffer to these reagents, as they are ready

for use. These reagents should be stored at 4°C (do not refreeze).

*NOTE: After reconstitution the standards must be further diluted prior to performing the assay.

Plate Set Up

There is no specific pattern for using the wells on the plate. However, it is necessary to have some wells (at

least two) designated as absorbance blanks (containing 200 μL of Assay Buffer or water). The absorbance of

these wells must then be subtracted from the absorbance measured in all the other wells. Standard curves for

nitrate and nitrite must also be included. If you plan to measure only total NO products (nitrate + nitrite), only

the nitrate standard curve is required. If only nitrite is being measured, then only the nitrite standard curve is

needed. The wells for the standard curves have been designated (as in A1-H2) in the instructions in Plate

Layout. However, these standard curves can be placed in any wells you choose. The remaining wells on the

plate can then be used for the assay of your samples. We suggest you record the contents of each well on the

template sheets provided (See Plate Layout).

This kit provides sufficient cofactors and reagents to run two 96-well plates measuring total NO (NO2- + NO3

-)

in all the wells. If you wish to test some samples for NO2- only (where reductase and cofactors are not required),

there is sufficient Griess Reagent R1 and R2 to run a third 96-well plate of nitrite determinations. All three

plates are supplied with this kit.


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Sample Preparation

The kit has been validated in urine, culture media, and plasma. No sample purification from these sources is

necessary other than some special instructions as described below. Store samples at -20°C or -80°C after

collection.

Urine samples - Urine can be used directly after dilution to the proper concentration in Assay Buffer.

Urine contains relatively high levels of nitrate (200-2,000 µM), so dilutions of approximately 1:10-1:50

may be necessary.

Culture Media - Some types of tissue culture media contain very high nitrate levels (i.e., RPMI 1640).

These types of media should not be used for cell culture if the goal of an experiment is to measure small

changes in nitrate levels. Cellular nitrate/nitrite production can be quantitated by subtracting the level ofnitrate/nitrite present in the media (in the absence of cells) from the total nitrate/nitrite level present during

cell growth. The effect of media components on color development can be assessed by making a Nitrite

Standard curve in the presence of a fixed volume of the culture media (40 µL works well) and comparing

it to a Nitrite Standard curve made in buffer alone.

Plasma and serum samples - Ultrafilter plasma or serum samples through a 10 or 30 kDa molecular

weight cut-off filter using a commercially available centrifuge or microfuge ultrafiltration device. The filters,

supplied through Amicon or Millipore, should be pre-rinsed with UltraPure water prior to ultrafiltration of

serum or plasma. Ultrafiltration will reduce background absorbance due to the presence of hemoglobin

and improve color formation using the Griess Reagents. Assay for nitrate and/or nitrite using a maximum

of 40 µL of the filtrate. The conversion of nitrate to nitrite requires three hours for completion.

Heparinized plasma may form a precipitate upon addition of Griess Reagent R1, thus making the sample

unusable for analysis. Citrate or EDTA are recommended as anticoagulants for plasma preparation.

Tissue homogenates - Homogenize the sample in PBS, pH 7.4, and centrifuge at 10,000 x g for 20

minutes. Ultracentrifuge the supernatant solution at 100,000 x g for 30 minutes (Centrifugation at 100,000

x g is optional, but will increase filtration rates). Ultrafilter using a 10 or 30 kDa molecular weight cut-off

filter using a commercially available centrifuge or microfuge ultrafiltration device. The filters, supplied

through Amicon or Millipore, should be pre-rinsed with UltraPure water prior to ultrafiltration. Assay the

sample for nitrate and/or nitrite using a maximum of 40 µL of the filtrate. The conversion of nitrate to

nitrite requires three hours for completion.

Assay Procedure

Measurement of Nitrate + Nitrite

Preparation of nitrate standard curve

A nitrate standard curve must be performed in order to quantitate sample nitrate + nitrite concentrations.

In a clean test tube place 0.9 mL of Assay Buffer. To this, add 0.1 mL of reconstituted nitrate standard


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and vortex. The concentration of this stock standard is 200 µM. Use this standard (200 µM) for the

preparation of the nitrate standard curve as described below. The standard curve for nitrate is prepared

by addition of reagents to the plate wells in the following way:

Well Nitrate Standard (μL) Assay Buffer (μL) Final Nitrate Concentration* (μM)

A1, A2 0 80 0

B1, B2 5 75 5

C1, C2 10 70 10

D1, D2 15 65 15

E1, E2 20 60 20

F1, F2 25 55 25

G1, G2 30 50 30

H1, H2 35 45 35

Table 1. Nitrate standard concentrations

*The concentration is calculated for the final 200 µL assay volume after addition of the Griess Reagents

Preparation of Samples for Total Nitrate + Nitrite Measurement

Samples containing nitrate (with or without nitrite) can be assayed by addition of up to 80 µL (40 µL with

plasma or serum) of sample per well and should be done in triplicate. When using less than 80 µL of

sample, the volume must be adjusted to 80 µL by addition of the appropriate amount of Assay Buffer.

When necessary, dilution of samples should be done using the Assay Buffer solution. In the event that

the approximate concentration of nitrate or nitrite is completely unknown, we recommend that severaldifferent dilutions of the sample be made.

The absorbance of the samples should be between 0.05 and 1.2 absorbance units, since the plate reader

will give the most accurate values when the absorbance is in this range. In addition, high absorbance

values imply high nitrate levels. Under these conditions, there may be incomplete conversion of nitrate to

nitrite. The detection limit of the assay is approximately 1 µM nitrite. When using 80 µL of sample, this

translates into 2.5 µM nitrate in the original sample.

Performing the Assay

1. Add 200 µL of water or Assay Buffer to the blank wells. Do not add any other reagents to these wells.

2. Add up to 80 µL of sample or sample dilutions to the wells in a pattern you choose. The final volume must

be adjusted to 80 µL using the Assay Buffer solution. NOTE: Plasma samples should be assayed with no

more than 40 µL when undiluted samples are used (Samples which have been diluted 1:2 or greater can

use up to 80 μ L in the assay). Caution should be taken when pipetting plasma samples to ensure no

bubbles enter to the well as this will lead to erroneous results.

3. Add 10 µL of the Enzyme Cofactor Mixture to each of the wells (standards and unknowns).

4. Add 10 µL of the Nitrate Reductase mixture to each of the wells (standards and unknowns).

5. Cover the plate with the plate cover and incubate at room temperature for one hour. NOTE: This

incubation time should be increased to two hours when assaying tissue culture medium, and increased to


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three hours when assaying plasma or tissue nitrate + nitrite concentrations. It is not necessary to shake

the plate during incubation.

6. After the required incubation time, add 50 µL of Griess Reagent R1 to each of the wells (standards and

unknowns).

7. Immediately add 50 µL of Griess Reagent R2 to each of the wells (standards and unknowns).

8. Allow the color to develop for 10 minutes at room temperature. It is not necessary to cover the plate.

NOTE: The 10 minute incubation is optimal for color development. However, if the plate has been left to

develop for longer time periods the data is still valid, provided the Griess reagents have been added to

the standard curve and unknowns at the same time. Developing the standard curve at the same time as

the unknowns ensures the presence of an accurate control.

9. Read the absorbance at 540 nm or 550 nm using a plate reader.

Measurement of Nitrite

Preparation of Nitrite standard curve

Nitrite concentrations can be measured directly by performing the assay in the absence of substrate or

enzymes. In a clean test tube place 0.9 mL of Assay Buffer. To this, add 0.1 mL of reconstituted nitrite

standard and vortex. Use this diluted standard (200 µM) for the preparation of the nitrite standard curve

as described below. The nitrite standard curve is prepared as follows:

Well Nitrite Standard (μL) Assay Buffer* (μL) Final Nitrite Concentration** (μM)

A1,A2 0 100 0

B1,B2 5 95 5

C1,C2 10 90 10

D1,D2 15 85 15

E1,E2 20 80 20

F1,F2 25 75 25

G1,G2 30 70 30

H1,H2 35 65 35

Table 2. Nitrite standard concentrations

*UltraPure water can also be used

**The concentration is calculated for the final 200 µL assay volume after addition of the Griess reagents.

Measurement of sample nitrite

Measurement of samples with unknown nitrite concentrations can be done using up to 100 µL of sample.

When using less than 100 µL of sample for nitrite determination, the volume must be adjusted to 100 µL

using Assay Buffer or water. Samples can be diluted in water or Assay Buffer. Once again it is best to

keep the absorbance of the sample at approximately 0.05-1.2. When using 100 µL of sample the

detection limit for nitrite is approximately 2 µM in the original sample.


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Performing the Assay

1. Add 200 μL of water or Assay Buffer to the blank wells. Do not add any other reagents to these wells.

2. Add up to 100 μL of sample to the chosen wells. When using less than 100 μL be sure to adjust the

volume to 100 μL using Assay Buffer or water.

3. Add 50 μL of Griess Reagent R1 followed by addition of 50 μL Griess Reagent R2 to each of the wells

(standards and unknowns).

4. Allow the color to develop for 10 minutes.

5. Measure the absorbance at 540 or 550 nm.


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Data Analysis

Calculation of Results

Subtract the blanks

Average the absorbance value of the blank wells and subtract this from the absorbance values of all the other

wells.

Plotting the standard curves

Make a plot of absorbance at 540-550 nm as a function of nitrate OR nitrite concentration. The nitrate standard

curve is used for determination of total nitrate + nitrite concentration, whereas the nitrite standard curve is used

for the determination of nitrite alone. In theory these two standard curves should be identical however, in

practice a small discrepancy often occurs.

Determination of sample nitrate or nitrite concentrations


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Figure 5. Typical standard curves

Performance Characteristics

Precision

The inter-assay coefficient of variation is 3.4% (n=5).

The intra-assay coefficient of variation is 2.7% (n=84).

Sensitivity

When using the maximum amount of sample for the nitrate/nitrite assay (80 µL), the detection limit is 2.5

µM. The detection limit for plasma is higher since only 40 µL of sample can be used. For the nitrite assay

a maximum volume of 100 µL can be used. In this case the detection limit is approximately 2.0 µM.

Interferences

Antioxidants will interfere with the color development reaction. Azide, ascorbic acid, dithiothreitol, and

mercaptoethanol will interfere with color development when present at concentrations as low as 100 μM.

Alkyl amines, most sugars, lipids, or amino acids (except those containing thiol groups) do not interfere.4

Phosphate concentrations greater than approximately 50 mM will interfere with the conversion of nitrate

to nitrite.


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Resources

Troubleshooting

Problem Possible Causes Recommended Solutions

Erratic values; dispersion of A. Poor pipetting/technique A. Be careful not to splash the

duplicates/triplicates B. Bubble in the well(s) contents of the wells

B. Carefully tap the side of the

plate with your finger to

remove bubbles

No color development in nitrate

standard curve

Cofactors or enzymes (or both) not

added.

You will need to do a new standard

curve. If you have not added one

of these reagents to the samplewells, you will need to repeat the

experiment.

References

1. Moncada, S. The L-arginine: nitric oxide pathway. Acta Physiol. Scand. 145, 201-227 (1992).

2. Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB Journal 6, 3051-3064 (1992).

3. Green, L.C., Wagner, D.A., Glogowski, J., et al. Analysis of nitrate, nitrite, and [15N] nitrate in biological

fluids. Anal. Biochem. 126, 131-138 (1982).

4. Nims, R.W., Darbyshire, J.F., Saavedra, J.E., et al. Colorimetric methods for the determination of nitric

oxide concentration in neutral aqueous solutions. Methods 7, 48-54 (1995).


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Plate Layout

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2 S t a n d a r d A

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A B C D E F G H

pedoman assay kit no ka1342

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